Funding provided in the first cycle of this grant helped to establish at Harvard Medical School the Drosophila RNAi Screening Center (DRSC). The purpose of the facility was to provide the community with a unique nfrastructure and expertise for high-throughput RNAi screens (RNAi HTS) in Drosophila cell lines using a genome-wide collection of double stranded RNAs (dsRNAs). The DRSC has been a resounding success. In less than 3 years, 75 applications from 43 different institutions have been approved by the center, 47 screens have been completed and 11 primary papers detailing the screens have been already published. The vast experience gained during these first years has been instrumental in shaping our view on how to build upon our initial goals and expand the scope and technology of RNAi HTS in Drosophila. To face those new challenges and develop the DRSC into an integrated center for technology transfer in functional genomics, we propose the following: First, the off-target effects observed with long dsRNAs will be addressed through a re-design of our library and the ability to offer two or more dsRNAs to confirm the specificity of RNAi phenotypes. Second, we will implement a number of key improvements to expand the range of functional screens available at the DRSC. We will offer sub-collections of dsRNAs against a variety of large gene families such as transcription factors or cytoskeletal proteins for more focused screens, and add miRNA and cDNA constructs over-expression libraries for gain of function studies in Drosophila cell- based assays. Third, to meet the growing demand on performing more sophisticated screens with greater biological relevance, we have added the ability to perform RNAi HTS in primary Drosophila cell cultures and have acquired an ultra high-throughput confocal, imaging platform that will be ideal for high content imaging screens. Fourth, we address the need of screeners to handle vast amount of data and the challenge to analyze them by planning a major upgrade of our database and developing powerful computational approaches for data analysis and data mining. Lastly, we provide two practical solutions to validate the results of the primary RNAi screen: the ability to perform focused RNAi screens in mammalian cells with siRNAs targeting human orthologs of genes identified in the Drosophila screen, and the generation of custom short hairpin RNA transgenic flies for rapid in vivo validation.